A specific and sensitive homologous radioimmunoassay has been developed for equine prolactin, suitable for measuring prolactin concentrations in serum of horses. The sensitivity of the assay ranged from 0.4 to 0.6 ng/ml and the intra- and inter-assay coefficients of variation averaged 6.9 and 15.4%, respectively, for five doses of hormone. Cross-reactivity with other mammalian and nonmammalian prolactins and growth hormones was less than 20 and 0.3%, respectively. Cross-reactivity with equine growth hormone was less than 0.07%. Equine serum and pituitary extracts showed parallel dilution-response curves with equine prolactin. The percentage recovery of exogenous equine prolactin in serum was 89%. Preliminary analysis of several physiological samples (stallions, pregnant, and nonpregnant mares) yielded values from 0.6 to 12.0 ng/ml.